Α5 Integrin Subunit Research Articles

Impaired integrin α5 /β1 -mediated hepatocyte growth factor release by stellate cells of the aged liver.

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α5/β1 is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and β1 decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α5 and β1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin β1 remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α5/β1 in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration.

Expression and distribution of CD151 as a partner of alpha6 integrin in male germ cells

The physiological importance of CD151 tetraspanin is known from somatic cells and its outside-in signalling through integrins was described. In male germ cells, two tetraspanins, CD9 and CD81, are involved in sperm-egg membrane fusion, and similarly to integrins, they occupy characteristic regions. We report here on a newly discovered presence of CD151 in sperm, and present its expression and distribution during spermatogenesis and sperm transition during the acrosome reaction. We traced CD151 gene and protein expression in testicular cell subpopulations, with strong enrichment in spermatogonia and spermatids. The testicular and epididymal localization pattern is designated to the sperm head primary fusion site called the equatorial segment and when compared to the acrosome vesicle status, CD151 was located into the inner acrosomal membrane overlying the nucleus. Moreover, we show CD151 interaction with α6 integrin subunit, which forms a dimer with β4 as a part of cis-protein interactions within sperm prior to gamete fusion. We used mammalian species with distinct sperm morphology and sperm maturation such as mouse and bull and compared the results with human. In conclusion, the delivered findings characterise CD151 as a novel sperm tetraspanin network member and provide knowledge on its physiology in male germ cells.

Heat Shock Cognate Protein 70 Enhanced Integrin β1 Mediated Invasion in Cancer Cells.

PurposeGlioblastoma is one of the most common malignant cancers worldwide. In our previous work, we have shown that heat shock cognate protein 70 (Hsc70) functions as a positive growth regulator in glioma. We investigated the role of Hsc70 in integrin β1 mediated invasion of glioma cells.MethodsIn order to investigate whether the down-regulation of Hsc70 would affect the expression of integrin β1 subunit, HeLa cells were transiently transfected with Hsc70-AS or pcDNA3.0 vectors and the down-regulation of Hsc70 was confirmed by Western blotting. Human brain glioma U87 cells were stably transfected with Hsc70-AS or pcDNA3.0 vectors to further elucidate the relationship between Hsc70 and integrin β1 in human glioma cells. Cellular localization of integrin β1 was detected using immunofluorescence confocal microscopy analysis.ResultsHere we reported that down-regulation of the expression of Hsc70 in U87 cells by transfection with antisense cDNA specifically increased the expression of cell surface integrin β1 without changing its mRNA. Meanwhile, the integrin β1 125-kD mature form increased while 105-kD precursor form decreased when Hsc70 was down-regulated. Mechanically, the U87 cells transfected with antisense cDNA of Hsc70 decreased the Golgi localization of integrin β1, strengthened its interaction with integrin α5 subunit, and enhanced the adhesion ability to fibronectin (FN) and the phosphorylation level of focal adhesion kinase (FAK).ConclusionOverall, these results suggested that the down-regulation of Hsc70 expression could promote the expression of cell surface integrin β1 and subsequently inhibit glioma invasion phenotype.

A Comprehensive Study of Human Integrins in Pediatric Lymphoblastic Leukemia Supports a Role of CD49f (Integrin α6) in the Localization to Bone Marrow but Not Spinal Fluid

Introduction: The overall survival of children with ALL (Acute Lymphoblastic Leukemia) has improved markedly over the last decade, yet relapse occurs in about 20% of patients. The spread of leukemic blasts to the Central Nervous System (CNS) and increased resistance to therapy due to cell adherence within the Bone Marrow (BM) or CNS constitutes major challenges to treatment. For these reasons, adhesion molecules governing the homing and adhesion of leukemic cells are perceived to be of extraordinary importance, both as potential biomarkers and therapeutic targets. Integrins constitute a large family of heterodimeric receptors composed of alpha and beta subunits, which play important roles during homing and migration of normal leucocytes by facilitating adhesion to both stromal cells and components of the extracellular matrix. Increased expression of CD49d (integrin subunit α4) is a marker for adverse prognosis in ALL and recently, CD49f (integrin subunit α6) was shown to facilitate metastasis of ALL xenografts to the central nervous system in mice (Yao H et al., Nature 2018). Methods Previous studies of integrins in BCP-ALL have focused on individual alpha integrins in xenograft models and were based on limited numbers of clinical samples. The present study was based on a large number of Danish pediatric BCP-ALL patients stratified between 2012-2018 using Minimal Residual Disease (MRD) according to the Nordic NOPHO-2008 protocol (Toft N et al., Eur J Haematol 2013). Diagnostic BM samples were subjected to flowcytometric analysis (FCM) of CD49f (n=246) and CD49d (n=135), using a backbone of lineage-specific B-cell markers (CD45, CD10, CD19, CD20). Leukemic blasts were detected in Cerebrospinal Fluid (CSF) using high-sensitivity FCM and the following markers CD45, CD10, CD19, CD20, CD34 and CD38 (n = 246, with matching BM and CSF samples). Results Our data provided us with a unique possibility to identify the role of CD49d and CD49f with respect to minimal residual disease (MRD) at the end of induction therapy (day 29), which is considered the most important prognostic factor in paediatric lymphoblastic leukemia. We found that CD49f was more highly expressed in patients with MRD ≥ 0,1% at day 29 than patients with MRD < 0,1% (p = 0,01), whereas no difference was seen with respect to CD49d. We also investigated the correlation between white blood cell (WBC) and surface expression of CD49d and CD49f in diagnostic BM blasts with respect to different cytogenetic subtypes. A Kruskall-Wallis test showed that the expression varies according to genetic subtypes (p‹0.0001). We found that the expression of CD49d was highest among the high hyperdiploid and iAMP21 (intrachromosomal amplification of chromosome 21) patients, whereas the expression of CD49f was highest among the t12,21 and iAMP21 patients. Notably, the expression of CD49f was inversely correlated to WBC (r=0,17, p=0.01), which was most pronounced among the patients in the B-other cytogenetic subgroup defined as leukaemia that could not be classified into the existing cytogenetic groups (r=0,42, P‹0.0001). In case of strong adherence to BM, lower levels of leukemic blasts might be expected in circulation resulting in high MRD but low WBC. Therefore, both MRD and WBC data are consistent with a prominent adhesive role of CD49f within BM. In contrast, we found significantly lower CD49f surface expression in diagnostic BM samples in patients with leukemic blasts within CSF (p=0.0297). Conclusions: Recently, Yao et al. (2018) showed that ALL cells in circulation are unable to breach the blood-brain barrier in mice and instead employ CD49f (integrin α6) to migrate into the CNS along vessels that connect vertebral or calvarial bone marrow and the subarachnoid space. Potentially, this mechanism could account for cases of ALL relapse within CNS. Our work shows a strong association between high MRD and the expression of CD49f which is a function that would have been anticipated for CD49d due to previous works with ALL and CLL. Furthermore, we found significantly lower CD49f surface expression in leukemic blasts within the BM in patients with CSF involvement and therefore no support for the recently proposed role of CD49f in facilitating the spread of leukemic cells to the CNS. Disclosures No relevant conflicts of interest to declare.

Integrin-Mediated Adhesion to Extracellular Matrix Protein Fibronectin Drives Megakaryocytosis in JAK2V617F+ Primary Myelofibrosis

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in Primary Myelofibrosis (PMF). Myelofibrosis was long regarded as a bystander of disease in PMF. However, as cells have the ability to sense the surrounding ECM through integrin receptors, we examined the hypothesis that abnormal ECM in myelofibrosis, mediated by integrin activation, contributes to BM megakaryocytosis in JAK2V617F+ PMF. Due to its complex genetic landscape, modeling PMF in animals is challenging. While there is no ideal model to replicate the human disease, Vav1-hJAK2V617F transgenic mice (JAK2V617F+) (Xing S et al. Blood. 2008; 111:5109-5117) are most appropriate for our studies because they express the most frequent mutation in PMF and show megakaryocytosis in BM and a predictable development of myelofibrosis throughout the animal's life. Fibronectin (FN) is a dimeric ECM glycoprotein produced by a variety of cells in BM. FN secretion by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Analysis of myelofibrotic BM from JAK2V617F+ mice revealed elevated levels of FN in BM ECM. Integrins are heterodimeric cell adhesion receptors of α and β subunits that connect the cell cytoskeleton to ECM. Integrin engagement and subsequent signaling affect cell differentiation, proliferation, migration and survival. Integrins α5β1, α4β1, αIIbβ3, and αυβ3 are FN-binding integrins known to be expressed in megakaryocytes. Expression analysis detected significantly elevated expression of α5, αIIb, αυ and β3 subunits in CD41+ megakaryocytes fromJAK2V617F+ mice in vitro and in vivo. Expression of β1 integrin was unchanged in megakaryocytes fromJAK2V617F+ mice, but expression of 9EG7, an antibody clone that detects the high-affinity conformation of β1 integrin, was elevated in JAK2V617F+ CD41+ megakaryocytes. Levels of the high-affinity form of β1 integrin decrease in response to inhibition of α5 integrin function (using Hmα5-1 antibody), indicating a high degree of correlation between affinity to FN and β1 activation in megakaryocytes fromJAK2V617F+ mice. As suggested by expression studies, in-vitro differentiated JAK2V617F+ megakaryocytes showed increased affinity to FN in adhesion assays. To ascertain the contribution of adhesion to FN in development of megakaryocytosis, megakaryocytes were differentiated in vitro on FN, with and without the presence of α5 integrin inhibitory antibody Hmα5-1. Culture on FN had a positive effect on the number of CD41+ megakaryocytes after four days of culture, especially in JAK2V617F+ BM. Importantly, inhibition of α5 integrin decreased the number of CD41+ megakaryocytes in JAK2V617F+ BM to nearly wild-type levels. To determine the effect of in vivo inhibition of α5 integrin on megakaryocyte numbers, the antibody clone 5H10-27, or MFR-5, previously reported to be effective in vivo, was used. Effective targeting in vivo of BM megakaryocytes by the 5H10-27 antibody was confirmed using a fluorochrome-labelled secondary antibody specific to the isotype of the injected clone (Rat IgG2a, κ). The same method was used to determine minimum required dose and interval of administration. JAK2V617F+ mice were treated with three doses of 5H10-27 or isotype control antibody (1mg/Kg IV every 48 hours). Analysis of treated animals on day 5 detected a decrease in megakaryocyte α5 integrin expression in animals treated with 5H10-27 antibody relative to control isotype antibody-treated animals. Platelet counts in peripheral blood and percentage of BM CD41+ megakaryocytes were consistently, albeit non-significantly, lower in 5H10-27 antibody-treated than in isotype antibody-treated animals. Corroborating our findings in mice, analysis of megakaryocytes from patients carrying the JAK2V617F mutation revealed elevated cell surface expression of α5 integrin subunit and increased adhesion to FN, which was dampened by an anti-α5 integrin antibody (Sam-1). Our results uncovered a global de-regulation of FN-binding integrin expression in megakaryocytes carrying the JAK2V617F mutation and the central role of FN-α5 integrin in development of megakaryocytosis in PMF. These results challenge the current paradigm by bringing ECM and myelofibrosis from a bystander position to center stage in the pathology of PMF. Disclosures No relevant conflicts of interest to declare.

Integrin α5 subunit is required for the tumor supportive role of fibroblasts in colorectal adenocarcinoma and serves as a potential stroma prognostic marker.

The tumorigenesis of colorectal cancer (CRC) is a complicated process, involving interactions between cancer cells and the microenvironment. The role of α5 integrin subunit in CRC remains controversial, and previous studies mainly focused on cancer cells. Herein, we report an important role of α5 in stroma fibroblasts in the tumorigenesis of CRC. The expression of α5 was found to be located in colorectal tumor stroma rather than in epithelia cancer cells. Immunofluorescence colocalization and gene correlation analysis confirmed that α5 was mainly expressed in cancer‐associated fibroblasts (CAFs). Moreover, experimental evidence showed that α5 expression was required for the tumor‐promoting effect of fibroblast cells. In an in vivo xenograft nude mice model, α5 depletion in fibroblasts dramatically suppressed fibroblast‐induced tumor growth. In an in vitro cell coculture assay, α5 depletion or knockdown reduced the ability of fibroblasts to promote cancer cell migration and invasion compared with wild‐type fibroblasts; moreover, we observed that the expression and assembly of fibronectin were downregulated after α5 depletion or knockdown in fibroblasts. Analysis of the RNA‐Seq data of the Cancer Genome Atlas cohort revealed that high expression of ITGA5 (α5 integrin subunit) was correlated with poor overall survival in colorectal adenocarcinoma, which was further confirmed by immunohistochemistry in an independent cohort of 355 patients. Thus, our study identifies α5 integrin subunit as a novel stroma molecular marker for colorectal adenocarcinoma, offers a fresh insight into colorectal adenocarcinoma progression, and shows that α5 expression in stroma fibroblasts underlies its ability to promote the tumorigenesis of colorectal adenocarcinoma.

P11.59 Integrin a5 heterogeneous expression in glioblastoma is related to glioma stem cell subpopulations

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive primary brain tumor. Treatment failure and recurrence are explained in part by tumoral heterogeneity. Our previous results showed that the integrin α5β1 is implicated in GBM aggressiveness and represents a relevant therapeutic target. Recently, we observed intra- and inter-tumor heterogeneity of integrin α5β1 expression. Heterogeneity may be linked to different glioma stem cell populations. MATERIAL AND METHODS Ten glioma stem cell lines were grown as neurospheres in stem cell medium and their differentiation was induced by serum and/or ATRA. Two cell lines (NCH421k and NCH644) were selected and were modified by depletion (CrisprCas9) or transfection of the α5 integrin gene. Polyclonal lines and individual clones were analyzed phenotypically in vitro, before and after differentiation, and in vivo in orthotopic xenografts of 2x104 cells in nude mice. TCGA datasets were used to validate the heterogeneous expression of α5 integrin in GBM. RESULTS TCGA data validate that α5 integrin mRNA was only over-expressed in the mesenchymal subclass of GBM. Our results show that α5 integrin protein is not expressed in stem cell culture conditions. However, α5 integrin expression is induced after differentiation in only half of the cell lines supporting the notion of tumoral heterogeneity of glioma stem cells. Interestingly, single cell-derived clone evaluation showed that intra-tumoral stem cell heterogeneity also exists at the level of α5 protein expression. When glioma stem cells are programmed or transduced to express α5 integrin, differentiated cells became more aggressive. Notably, they acquired a fibronectin-dependent motility and a proliferative phenotype. Interestingly, integrin α5 remained expressed in secondary stem cells obtained after dedifferentiation. The in vivo assays suggested that glioma stem cells, programmed to express the integrin, were prone to form larger tumors. CONCLUSION Our data support the hypothesis that some glioma stem cells are programmed to express the α5 integrin subunit in their differentiated progeny to form a more aggressive tumor. They add new evidences that both cell populations may be considered for new therapeutic strategies against GBM.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2β1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

Establishment of a Murine Pro-acinar Cell Line to Characterize Roles for FGF2 and \u03b13\u03b21 Integrins in Regulating Pro-acinar Characteristics

Radiation therapy for head and neck cancers results in permanent damage to the saliva producing acinar compartment of the salivary gland. To date, a pure pro-acinar cell line to study underlying mechanisms of acinar cell differentiation in culture has not been described. Here, we report the establishment of a pro-acinar (mSG-PAC1) and ductal (mSG-DUC1) cell line, from the murine submandibular salivary gland (SMG), which recapitulate developmental milestones in differentiation. mSG-DUC1 cells express the ductal markers, keratin-7 and keratin-19, and form lumenized spheroids. mSG-PAC1 cells express the pro-acinar markers SOX10 and aquaporin-5. Using the mSG-PAC1 cell line, we demonstrate that FGF2 regulates specific steps during acinar cell maturation. FGF2 up-regulates aquaporin-5 and the expression of the α3 and α6 subunits of the α3β1 and α6β1 integrins that are known to promote SMG morphogenesis and differentiation. mSG-DUC1 and mSG-PAC1 cells were derived from genetically modified mice, homozygous for floxed alleles of the integrin α3 subunit. Similar to SMGs from α3-null mice, deletion of α3 alleles in mSG-PAC1 cells results in the up-regulation of E-cadherin and the down-regulation of CDC42. Our data indicate that mSG-DUC1 and mSG-PAC1 cells will serve as important tools to gain mechanistic insight into salivary gland morphogenesis and differentiation.

Osteoblasts grown on microroughened titanium surfaces regulate angiogenic growth factor production through specific integrin receptors

Cellular attachment and response to biomaterials are mediated by integrin receptor binding to extracellular matrix proteins adsorbed onto the material surface. Osteoblasts interact with their substrates via several integrin complexes including fibronectin-binding α5β1 and collagen-binding α1β1 and α2β1. Knockdown of α2 or β1 integrin subunits inhibits the production of factors that promote an osteogenic microenvironment, including osteocalcin, osteoprotegerin, and TGFβ1. Osteoblasts also secrete several angiogenic growth factors such as VEGF-A (VEGF165), FGF-2, and angiopoietin 1, which are regulated by titanium surface topography and surface energy. Here, we examined whether signaling through integrin receptor complexes regulates production and secretion of angiogenic factors during osteoblast differentiation on microtextured Ti surfaces. To do this, integrin subunits α1, α2, α5, and β1 were stably silenced in MG63 osteoblast-like cells cultured on grit-blasted/acid-etched hydrophobic Ti (SLA) or on hydrophilic SLA (modSLA). VEGF-A production increased in response to Ti surface topography and energy in integrin α2, α5, and β1 silenced cells but decreased in α1-silenced cells. FGF-2 decreased on modSLA substrates in both α1 and α2-silenced cells but was unchanged in response to silencing of either α5 or β1. In integrin α1, α2, and β1-silenced cells, Ang-1 increased on modSLA but α5-silencing did not affect Ang-1 production during surface mediated differentiation. These results suggest that signaling through specific integrin receptor complexes during osteoblast differentiation on microstructured Ti substrates, regulates the production of angiogenic factors by those cells, and this is differentially regulated by surface hydrophilicity. Statement of SignificanceSuccessful implantation of synthetic biomaterials into bone depends on the biological process known as osseointegration. Osseointegration is a highly regulated communication of cells that orchestrates the migration of progenitor cells towards the implant site and promotes the deposition and mineralization of extracellular matrix proteins within the implant microenvironment, to tightly join the implant to native bone. In this process, angiogenesis functions as the initiation site of progenitor cell migration and is necessary for matrix deposition by providing the necessary nutrients for bone formation. In the present study, we show a novel regulation of specific angiogenic growth factors by integrin receptor complexes. This research is important to develop biomaterials that promote and maintain osseointegration through proper vascularization and prevent implant failure in patients lacking sufficient angiogenesis.

Crystal structures of Rea1-MIDAS bound to its ribosome assembly factor ligands resembling integrin\u2013ligand-type complexes

The Rea1 AAA+-ATPase dislodges assembly factors from pre-60S ribosomes upon ATP hydrolysis, thereby driving ribosome biogenesis. Here, we present crystal structures of Rea1-MIDAS, the conserved domain at the tip of the flexible Rea1 tail, alone and in complex with its substrate ligands, the UBL domains of Rsa4 or Ytm1. These complexes have structural similarity to integrin α-subunit domains when bound to extracellular matrix ligands, which for integrin biology is a key determinant for force-bearing cell–cell adhesion. However, the presence of additional motifs equips Rea1-MIDAS for its tasks in ribosome maturation. One loop insert cofunctions as an NLS and to activate the mechanochemical Rea1 cycle, whereas an additional β-hairpin provides an anchor to hold the ligand UBL domains in place. Our data show the versatility of the MIDAS fold for mechanical force transmission in processes as varied as integrin-mediated cell adhesion and mechanochemical removal of assembly factors from pre-ribosomes.

Conjugation of the T1 sequence from CCN1 to fibrin hydrogels for therapeutic vascularization.

Hydrogel matrices with angiogenic properties are much desirable for therapeutic vascularization strategies, namely to provide vascular supply to ischemic areas, transplanted cells, or bioengineered tissues. Here we report the pro-angiogenic effect of fibrin (Fb) functionalization with the T1 sequence from the angiogenic inducer CCN1, forseeing its use in the injured brain and spinal cord. Fibrin functionalization with 40 μM of T1 peptide effectively improved cellular sprouting of human brain microvascular endothelial cells (hCMEC/D3) in the absence of vascular endothelial growth factor (VEGF), without impacting the viscoelastic properties of Fb, cell viability, or proliferation. The pro-angiogenic effect of immobilized T1 was potentiated in the presence of VEGF and partially mediated through α6β1 integrin. The tethering of T1 also enhanced sprouting of human cord blood-derived outgrowth endothelial cells (OEC). Still, to elicit such effect, a higher input T1 concentration was required (60 μM), in line with the lower protein levels of α6 and β1 integrin subunits found in OEC comparing to hCMEC/D3, prior to embedment in Fb gel. Finally, the ability of T1-functionalized Fb in inducing cappilary invasion in vivo was assessed using the CAM assay, which evidenced a significant increase in the number of newly formed vessels at sites of implantation of T1-functionalized Fb, in the absence of soluble angiogenic factors. Overall these results demonstrate the potential of T1 peptide-presenting gels for use in therapeutic vascularization approaches. Considering T1 neurite-extension promoting capability and pro-angiogenic properties, T1-functionalized Fb hydrogels are particularly promising for application in the injured central nervous system.

Identification of differentially expressed genes in pancreatic ductal adenocarcinoma and normal pancreatic tissues based on microarray datasets.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor with rapid progression and poor prognosis. In the present study, 11 high‑quality microarray datasets, comprising 334 tumor samples and 151 non‑tumor samples from the Gene Expression Omnibus, were screened, and integrative meta‑analysis of expression data was used to identify gene signatures that differentiate between PDAC and normal pancreatic tissues. Following the identification of differentially expressed genes (DEGs), two‑way hierarchical clustering analysis was performed for all DEGs using the gplots package in R software. Hub genes were then determined through protein‑protein interaction network analysis using NetworkAnalyst. In addition, functional annotation and pathway enrichment analyses of all DEGs were conducted in the Database for Annotation, Visualization, and Integrated Discovery. The expression levels and Kaplan‑Meier analysis of the top 10 upregulated and downregulated genes were verified in The Cancer Genome Atlas. A total of 1,587 DEGs, including 1,004 upregulated and 583 downregulated genes, were obtained by comparing PDAC with normal tissues. Of these, hematological and neurological expressed 1, integrin subunit α2 (ITGA2) and S100 calcium‑binding protein A6 (S100A6) were the top upregulated genes, and kinesin family member 1A, Dymeclin and β‑secretase 1 were the top downregulated genes. Reverse transcription‑quantitative PCR was performed to examine the expression levels of S100A6, KRT19 and GNG7, and the results suggested that S100A6 was significantly upregulated in PDAC compared with normal pancreatic tissues. ITGA2 overexpression was significantly associated with shorter overall survival times, whereas family with sequence similarity 46 member C overexpression was strongly associated with longer overall survival times. In addition, network‑based meta‑analysis confirmed growth factor receptor‑bound protein 2 and histone deacetylase 5 as pivotal hub genes in PDAC compared with normal tissue. In conclusion, the results of the present meta‑analysis identified PDAC‑related gene signatures, providing new perspectives and potential targets for PDAC diagnosis and treatment.

Identification of biomarkers and their functions in dasatinib-resistant pancreatic cancer using bioinformatics analysis.

Dasatinib is a tyrosine kinase inhibitor, which inhibits tumor proliferation by blocking SRC pathways and is considered as a potential treatment of various epithelial neoplasms, including pancreatic cancer. However, dasatinib efficacy is largely limited due to drug resistance. In the present study, bioinformatics strategies were used to investigate the potential mechanisms of dasatinib-resistance in pancreatic cancer. The gene expression profiles of the Panc0403, Panc0504, Panc1005 (dasatinib-sensitive), SU8686, MiaPaCa2 and Panc1 (acquired dasatinib-resistant) cell lines were obtained from the gene expression omnibus database. The differentially expressed genes (DEGs) were then selected using R software. In addition, gene ontology (GO) and pathway enrichment analysis were performed through the Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction (PPI) network was constructed and analyzed to determine the hub genes using the Search Tool for the Retrieval of Interacting Genes database. A total of 472 DEGs, including vimentin, transmembrane 4 l six family member 18 and S100 calcium binding protein P, were identified. Enrichment analysis by GO function demonstrated that DEGs were associated with extracellular components, signal regulation and binding factors. The analysis of the Kyoto Encyclopedia of Genes and Genomes demonstrated that several adenocarcinoma pathways were enriched, including the phosphoinositide 3-kinases/protein kinase B and mitogen-activated protein kinase signaling pathways. Some hub genes were highlighted following the PPI network construction, including Rac family small GTPase 1, laminin subunit α3, integrin subunit β4, integrin subunit α2, collagen type VI α1 chain, collagen type I α2 chain, arrestin β1 and synaptotagmin 1, which may be associated with pancreatic adenocarcinoma prognosis. A total of five out of eight hub genes were highly associated with the overall survival rate (P<0.05). In conclusion, the present study reported novel insights into the mechanisms of dasatinib resistance. Identification of these hub genes may be considered as potential novel treatment targets for dasatinib-resistance in pancreatic cancer.

Deciphering the Mammary Stem Cell Niche: A Role for Laminin-Binding Integrins.

SummaryIntegrins, which bind laminin, a major component of the mammary basement membrane, are strongly expressed in basal stem cell-enriched populations, but their role in controlling mammary stem cell function remains unclear. We found that stem cell activity, as evaluated in transplantation and mammosphere assays, was reduced in mammary basal cells depleted of laminin receptors containing α3- and α6-integrin subunits. This was accompanied by low MDM2 levels, p53 stabilization, and diminished proliferative capacity. Importantly, disruption of p53 function restored the clonogenicity of α3/α6-integrin-depleted mammary basal stem cells, while inhibition of RHO or myosin II, leading to decreased p53 activity, rescued the mammosphere formation. These data suggest that α3/α6-integrin-mediated adhesion plays an essential role in controlling the proliferative potential of mammary basal stem/progenitor cells through myosin II-mediated regulation of p53 and indicate that laminins might be important components of the mammary stem cell niche.

Implication of integrin α2β1 in anoikis of SK-Mel-147 human melanoma cells: a non-canonical function of Akt protein kinase.

Suppression of anoikis, a kind of apoptosis caused by disruption of contacts between cell and extracellular matrix, is an important prerequisite for cancer cell metastasis. In this communication, we demonstrate that shRNA-mediated depletion of α2 integrin subunit induces anoikis and substantially decreases colony-forming potential in SK-Mel-147 human melanoma cells. Suppression of α2β1 upregulates the levels of pro-apoptotic protein p53 and cyclin-dependent kinase inhibitors p21 and p27. Concomitantly, we detected decrease in the levels of anti-apoptotic protein Bcl-2 and cell cycle regulator c-Myc. Moreover, depletion of α2β1 reduces the activity of protein kinase Erk, while increases activity of Akt kinase. Pharmacological inhibition of P3IK kinase, an upstream activator of Akt, greatly enhanced anoikis in control cells while reduced that in cells with decreased levels of α2β1. Of three isoforms of Akt, down-regulation of Akt1 greatly diminished anoikis of cells depleted of α2β1, while down-regulation of Akt2 and Akt3 sharply increased anoikis in these cells. These findings were supported by the data of pharmacological inhibition of the Akt isoforms. Our results demonstrate for the first time that anoikis induced by α2β1 integrin knockdown can be attenuated by Akt1 inhibition.

Addressing the Compartmentalization of Specific Integrin Heterodimers in Mouse Sperm.

Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction.

The effect of substrate bulk stiffness on focal and fibrillar adhesion formation in human abdominal aortic endothelial cells

Endothelial cell (EC) dysfunction contributes to atherosclerosis, which is associated with arterial stiffening and fibronectin (FN) deposition, by ECs and smooth muscle cells (SMCs). The effect of stiffness on the EC/FN interaction and fibrillar adhesion formation has been poorly studied. An in vitro model was prepared that included FN-coated polydimethylsiloxane (PDMS) films with similar hydrophobicity and roughness but distinct Young's modulus values, mimicking healthy (1.0 MPa) and atherosclerotic (2.8 MPa) arteries. Human aortic abdominal endothelial cells (HAAECs) seeded on 1.0 MPa PDMS films spread over time and reached their maximum surface area faster than on 2.8 MPa PDMS films. In addition, HAAECs appeared to organize focal adhesion more rapidly on 1.0 MPa PDMS films, despite the similar cell binding domain accessibility to adsorbed FN. Interestingly, we also observed up to a ~5-fold increase in the percentage of HAAECs that had a well-developed fibrillar adhesion on 1.0 MPa compared to 2.8 MPa PDMS films as verified by integrin α5 subunits, tensin, and FN staining. This variation did not affect EC migration. These results suggest that there are favourable conditions for FN matrix assembly by ECs in early atherosclerosis rather than at advanced stages. Our in vitro model will therefore be helpful to understand the influence of bulk stiffness on cells involved in atherosclerosis.

The Interaction between Laminin-332 and α3β1 Integrin Determines Differentiation and Maintenance of CAFs, and Supports Invasion of Pancreatic Duct Adenocarcinoma Cells.

Ranking among the most lethal tumour entities, pancreatic duct adenocarcinoma cells invade neighbouring tissue resulting in high incidence of metastasis. They are supported by tumour stroma fibroblasts which have undergone differentiation into cancer-associated fibroblasts (CAFs). Stiffness of cell substratum, cytokines, such as transforming growth factor-β (TGF-β), and stromal matrix proteins, such as laminin-332, are factors which promote CAF differentiation. In a spheroid culture system, differentiation of CAFs was analysed for laminin-332 production, laminin-binding integrin repertoire, adhesion and migration behaviour, and, in heterospheroids, for their interplay with the pancreatic duct adenocarcinoma AsPC-I cells. Our data reveal that CAFs produce laminin-332 thus contributing to its ectopic deposition within the tumour stroma. Moreover, CAF differentiation correlates with an increased expression of α3β1 integrin, the principal laminin-332-receptor. Beyond its role as novel CAF marker protein, integrin α3β1 crucially determines differentiation and maintenance of the CAF phenotype, as knock-out of the integrin α3 subunit reversed the CAF differentiated state. AsPC-I cells co-cultured in heterospheroids with integrin α3-deficient CAFs invaded less than from heterospheroids with wild-type CAFs. This study highlights the role of integrin α3β1 integrin-laminin-332 interaction of CAFs which promotes and sustains differentiation of CAFs and promotes carcinoma invasion.

Downregulation of TREM-like transcript-1 and collagen receptor α2 subunit, two novel RUNX1-targets, contributes to platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia.

Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by RUNX1. We aimed to identify RUNX1-targets involved in platelet function by combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (β)-1, which are involved in platelet aggregation and adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates TREML1 and ITGA2, and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of TREML1 and ITGA2 clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML.